Assay for diagnosing Streptococcus pneumoniae

ABSTRACT

Assays for detecting anti-streptococcal antibodies in biological samples using one or more streptococcal antigens are described herein. Various combinations of antigens may be used in the assays. For example, one or more of Ply, PhtD, PhtE, LytB and PcpA may be utilized. Additional streptococcal antigens may also be used. The assays may also be used in combination with assays that detect streptococcal nucleic acids.

RELATED APPLICATIONS

This application was filed under 35 U.S.C. §371, and claims priority toInternational Application No. PCT/CA2009/000119, filed Feb. 2, 2009,which claims priority to U.S. Ser. No. 61/063,376 filed Feb. 1, 2008,each of which are incorporated herein by reference in their entirety.

FIELD OF THE DISCLOSURE

Described herein are reagents and methods for diagnosing bacterialinfections.

BACKGROUND OF THE DISCLOSURE

Community-acquired pneumonia (CAP) is one of the most common pediatricinfections, a major cause of morbidity in industrialized countries, anda leading cause of mortality worldwide in children under five years ofage (Mulholland K, Lancet 2007; 370(9583): 285-9); Pio A, BWHO; 2003;81(4):298-300). CAP may be caused by multiple agents alone or incombination, including respiratory viruses, Streptococcus pneumoniae,Haemophilus influenzae, Chlamydia and Mycoplasma pneumoniae (Yin C CRespirology, 2003; 8(1):83-9; Chiang W C Respirology 2007; 12(2):254-61;Juven T PIDJ 2000; 19(4): 293-8). Etiological diagnosis is particularlychallenging in young children, in whom blood cultures usually remainnegative, bronchial secretions are rarely accessible, and frequentnasopharyngeal carriage limits the usefulness of microbial detection.Increasing the number of assays for pathogen detection neverthelessenhances the likelihood of the identification of a causal agent (WangPediatr Pulmonol 2008 feb 43(2)150-9; Nakayama E J Infect Chemother2007, 13(5):305-13), and recent studies using modern diagnostic toolshave repeatedly identified S. pneumoniae as a leading cause of CAP, bothin primary-care, and in hospital settings (Juven T PIDJ 2000; 19(4):293-8; Michelow I C; Pediatrics 2004; 113(4):701-7).

Protection against pneumococcal infection is essentially mediated byantibodies promoting opsonophagocytosis. Such antibodies may be directedagainst pneumococcal polysaccharides (PPS) or pneumococcal surfaceproteins (PSP). In the absence of pneumococcal immunization, suchantibodies are elicited by pneumococcal exposure, colonization and/orinfections. The lack of protective antibodies against pneumococcalantigens is thus at the basis of the vulnerability of infants and youngchildren to pneumococcal disease. Although certain PSPs have been usedin the serological diagnosis of pneumococcal infections, with excellentspecificities and positive predictive values, such studies havefrequently been limited by a low sensitivity in children. This waslargely attributed to higher colonization prevalence interfering withserological analyses (Scott, et al. Clin. Diagn. Lab. Immunol. 2005).

The etiological diagnosis of pneumococcal pneumonia remains challenging,particularly in young children. Microbial diagnosis based on bloodculture or DNA amplification has a high specificity but a lowsensitivity in children who are rarely bacteremic. In contrast, highrates of nasopharyngeal colonization limits the sensitivity of assaysdetecting bacteria and/of fragments thereof in nasopharynx or urinesamples (Dowell, S. F., Clin. Infect. Dis. 2000 32:824-825). The questfor a robust methodology of serological diagnosis of pneumococcalpneumonia has been met with many difficulties (Kanclerski, J ClinMicrobiol 1988; Korppi M, Eur J Clin Microbiol Infect Dis 2007).

Diagnosis of community-acquired pneumonia (CAP) is particularlychallenging in young children through traditional laboratory methods.There is a need in the field for reagents and methods useful foraccurately and quickly diagnosing CAP, especially in young children.This need relates to both individual diagnosis, which requires assaysproviding rapid results with high sensitivities and negative predictivevalues to avoid useless antibiotic administration, and inepidemiological studies requiring assays with high specificity andpositive predictive values for optimal case definition and adequatesensitivity to correctly estimate disease burden. Reagents and methodsfor accurately and quickly diagnosing CAP are described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Anti-PSP serum IgG antibodies at admission.

FIG. 2. Distribution of anti-PhtD IgG antibodies in children withpneumococcal or non-pneumococcal CAP.

FIG. 3. Distribution of anti-PhtE IgG antibodies in children withpneumococcal or non-pneumococcal CAP.

FIG. 4. Distribution of anti-Ply IgG antibodies in children withpneumococcal or non-pneumococcal CAP.

FIG. 5. Distribution of anti-PcPA IgG antibodies in children withpneumococcal or non-pneumococcal CAP.

SUMMARY OF THE DISCLOSURE

Assays for detecting anti-streptococcal antibodies in biological samplesusing one or more streptococcal antigens are described herein. Variousantigens alone or combinations of antigens may be used in these assays.For instance, the assay may be performed to detect antibodiesimmunoreactive to Ply and one or more of PhtD, PhtE, LytB and PcpA; PhtDand one or more of Ply, PhtE, LytB and PcpA; PhtE and one or more ofPly, PhtD, LytB and PcpA; LytB and one or more of Ply, PhtD, PhtE, andPcpA; and/or PcpA and one or more of Ply, PhtD, PhtE, and LytB. Theassays described herein may also be used with other streptococcalantigens in combination with one another and/or one or more of Ply,PhtD, PhtE, LytB, and/or PcpA. In this manner, sensitivity of the assayis increased and the negative predictive value is improved.

DETAILED DESCRIPTION

Provided herein are reagents and methods for accurately and quicklydiagnosing community-acquired pneumonia (CAP) are described herein. Theassays described herein overcome the low sensitivity of pneumococcalsurface protein (PSP)-based immunoassays in children due to thetraditional use of only one or a few immunogenic pneumococcal antigens.By detecting antibodies to a combination of bacterial antigens in theserum or other biological fluid of patient, a CAP diagnosis may be made.As shown herein, multiple PSPs were utilized as immunological probes toassess the pattern of humoral immunity driven by past exposure and acutepneumococcal infection in young children hospitalized for CAP. In oneembodiment, the antigens may be selected from pneumolysin (Ply), PhtD,PhtE, LytB, and/or PcpA. The sequences of these five pneumococcalsurface proteins are widely conserved across pneumococcal strains(>95-98%), allowing their use as markers of exposure to S. pneumoniae aswell as vaccine candidates.

Pneumolysin (Ply) is a cytolytic-activating toxin implicated in multiplesteps of pneumococcal pathogenesis, including the inhibition of ciliarybeating and the disruption of tight junctions between epithelial cells(Hirst et al. Clinical and Experimental Immunology (2004)). Severalpneumolysins are known and would be suitable in practicing the assaysdescribed herein including, for example GenBank Accession Nos. Q04IN8,P0C2J9, Q7ZAK5, and ABO21381, among others. In one embodiment, Ply hasthe amino acid sequence shown in SEQ ID NO.: 1.

PhtD polypeptides suitable for practicing the assays described hereininclude, for example, those of GenBank Accession Nos. AAK06760, YP816370and NP358501, among others. In one embodiment, PhtD has the amino acidsequence shown in SEQ ID NO.: 2.

PhtE polypeptides suitable for practicing the assays described hereininclude, for example, those of GenBank Accession Nos. AAK06761, YP816371and NP358502, among others. In one embodiment, PhtE has the amino acidsequence shown in SEQ ID NO.: 3.

LytB polypeptides suitable for practicing the assays described hereininclude, for example, those of GenBank Accession Nos. CAA09078,YP816335, ABJ55408, AAK19156, NP358461, and AAK75086, among others. Inone embodiment, LytB has the amino acid sequence shown in SEQ ID NOS.:4, 5 or 8.

PcpA was first cloned and characterized in 1998 as a choline-bindingprotein with a putative adhesion role (Sanchez-Beato A FEMS MicrobiologyLetters 164 (1998) 207-214). PcpA polypeptides suitable for practicingthe assays described herein include, for example, those of GenBankAccession Nos. CAB04758, YP817353, AAK76194, NP359536, ZP01835022, andZP01833419, among others. In one embodiment, PcpA has the amino acidsequence shown in SEQ ID NOS.: 6 or 7.

The antigens may be used in the assays described herein, either alone orin combination with one another. The use of single antigens or anycombination of antigens may be suitable for use in the assays describedherein provided the assay demonstrates the desired sensitivity andnegative predictive values. In certain embodiments, the use of acombination of antigens may result in a sensitivity of approximately,0.80, 0.85, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99,or 1.0 with a negative predictive value of approximately 0.80, 0.85,0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, or 1.0. Insome embodiments, the values are significant. Comparisons may beperformed and significance determined using any of the availablestatistical analysis tools, alone or in combination with one another,including, for example, student's T-test, chi-square test, Fisher'sexact test, analysis of variance (ANOVA), univariate statisticalanalyses, logistic regression analysis to calculate adjusted odds ratio(OR) and 95% confidence interval (CI). Conrols for any statisticallysignificant demographic variables that might function as confounders(gender, etc) may also be utilized. Differences between values aretypically considered significant at p<0.05 or p<0.01, for example. Otherstatistical analysis tools may also be used.

For instance, the assays may be performed to detect antibodiesimmunoreactive to only one of Ply, PhtD, PhtE, LytB or PcpA, withoutassaying for antibodies reactive to any other antigen. Alternatively,the assay may be performed to detect antibodies immunoreactive to morethan one of Ply, PhtD, PhtE, LytB and/or PcpA. For instance, the assaymay be performed to detect antibodies immunoreactive to Ply and one ormore of PhtD, PhtE, LytB and PcpA; PhtD and one or more of Ply, PhtE,LytB and PcpA; PhtE and one or more of Ply, PhtD, LytB and PcpA; LytBand one or more of Ply, PhtD, PhtE, and PcpA; and/or PcpA and one ormore of Ply, PhtD, PhtE, and LytB. The assay may also be performed toidentify antibodies immunoreactive to combinations of antigens such asPcpA and Ply; PcpA and PhtD; PcpA and PhtE; PcpA and LytB; PcpA, Ply,and PhtD; PcpA, Ply, PhtD, and PhtE; Ply, PhtD, PhtE, and LytB; PcpA,PhtD, and PhtE; PcpA, PhtD, PhtE, and LytB; PcpA, PhtE, and LytB; PcpA,Ply, PhtE, and LytB; PcpA, PhtD, and LytB; PcpA, Ply, and LytB; PcpA,Ply, PhtD, and LytB; PcpA, Ply, and PhtE; and/or, PcpA, PhtD, and PhtE.Other combinations will be apparent to one of skill in the art giventhis disclosure. The assays described herein may also be used with otherstreptococcal antigens in combination with one another and/or one ormore of Ply, PhtD, PhtE, LytB, and/or PcpA.

In certain embodiments, an isolated and purified PcpA protein orimmunologically reactive fragment thereof may be used for detecting apast infection or active infection by Streptococcus pneumoniae in asubject by detecting the binding of antibodies in a sample obtained froma subject to said isolated and purified PcpA antigen (e.g., protein orimmunologically reactive fragment thereof). In other embodiments, theisolated and purified PcpA antigen may be used with at least oneadditional antigen (e.g, PhtD, PhtE, LytB and/or Ply protein orimmunologically reactive fragment thereof). Thus, a method of detect thepresence of and/or diagnosing pneumoniae or infection by S. pneumoniaein a subject comprising detecting in a biological sample from saidsubject antibodies against one or more PcpA, PhtD, PhtE, LytB, and/orPly antigens, wherein the presence of antibodies that bind to theantigen(s) is indicative of infection, is provided. In certainembodiments, the method comprises contacting a biological sample derivedfrom a subject with the isolated or purified PcpA, PhtD, PhtE, LytB,and/or Ply antigens of S. pneumoniae for a time and under conditionssufficient for an antigen-antibody complex to form and then detectingthe formation of an antigen-antibody complex. Detection of theantigen-antibody complex may be achieved by detecting humanimmunoglobulin in the complex. In certain embodiments, detection of theantibody may be accomplished by contacting the antigen-antibody complexwith a “second” antibody that is immunologically reactive with humanimmunoglobulin (e.g., anti-human immunoglobulin antibody) for a time andunder conditions sufficient for the second antibody to bind to the humanimmunoglobulin in the complex and then detecting the bound anti-humanimmunoglobulin. It is preferred that the second antibody is labelledwith a detectable marker or reporter molecule.

A method is also provided for determining the response of a subjecthaving pneumonia or an infection by Streptococcus pneumoniae totreatment with a therapeutic compound. The method involves detectingantibodies against a PcpA antigen in a biological sample of the subjectafter treatment, wherein the amount of antibody detected is increased,unchanged, or decreased as compared to the amount of antibody detectablein a biological sample of the subject obtained prior to treatment, or tothat of a normal or healthy subject. In one embodiment, an unchanged ordecreased amount of antibody after treatment may indicate that thesubject is not responding to treatment. In another embodiment, anunchanged or decreased amount of antibody after treatment may indicatethat the subject is responding to treatment. In another embodiment, anincreased amount of antibody after treatment may indicate that thesubject is not responding to treatment. In another embodiment, anincreased amount of antibody after treatment may indicate that thesubject is responding to treatment. Treatment regimens may then beadjusted accordingly.

The antigens may be used in these assays as full-length polypeptides.However, the antigens may be related antigens such as, for example,fragments, variants (e.g., allelic, splice variants), orthologs,homologues, and derivatives (e.g., peptides, fusions) that retainreactivity to antibodies found in the biological sample. A fragment,variant, or derivative may have one or more sequence substitutions,deletions, and/or additions as compared to the subject sequence.Fragments or variants may be naturally occurring or artificiallyconstructed. In one embodiment, such related antigens may be preparedfrom the corresponding nucleic acid molecules. In preferred embodiments,the related antigens may have from 1 to 3, or from 1 to 5, or from 1 to10, or from 1 to 15, or from 1 to 20, or from 1 to 25, or from 1 to 30,or from 1 to 40, or from 1 to 50, or more than 50 amino acidsubstitutions, insertions, additions and/or deletions. Related antigensmay include peptides, which are typically a series of contiguous aminoacid residues having a sequence corresponding to at least a portion ofthe polypeptide from which it was derived. In preferred embodiments, apeptide may include about 5-10, 10-15, 15-20, 30-20, or 30-50 aminoacids.

Substitutions may be conservative, or non-conservative, or anycombination thereof. Conservative amino acid modifications to thesequence of an antigen (and/or the corresponding modifications to theencoding nucleotides) may produce a related antigen having functionaland chemical characteristics similar to those of parental antigen. Forexample, a “conservative amino acid substitution” may involve asubstitution of a native amino acid residue with a non-native residuesuch that there is little or no effect on the size, polarity, charge,hydrophobicity, or hydrophilicity of the amino acid residue at thatposition and, in particlar, does not result altered function of theantigen (e.g., does not cause decreased immunogenicity or reactivitywith antibodies in the biological sample). Suitable, exemplaryconservative amino acid substitutions are shown below:

Original Preferred Residues Exemplary Substitutions Substitutions AlaVal, Leu, Ile Val Arg Lys, Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser,Ala Ser Gln Asn Asn Glu Asp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, ArgArg Ile Leu, Val, Met, Ala, Phe, Norleucine Leu Leu Norleucine, Ile,Val, Met, Ala, Phe Ile Lys Arg, 1,4 Diamino-butyric Acid, Gln, Asn ArgMet Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Tyr Leu Pro Ala Gly SerThr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, SerPhe Val Ile, Met, Leu, Phe, Ala, Norleucine Leu

Fragments may include antigens with particular regions or domainsdeleted. It may be beneficial to delete such regions or domains where,for instance, the presence of those regions or domains will interferewith the use of the fragments in the assays described herein. Forinstance, when using PcpA, Ply, PhtD, PhtE or LytB, the cholinergicbinding region may be deleted. An exemplary fragment of PcpA protein isshown in SEQ ID NO.: 7.

In other embodiments, the antigen may include one or more fusionpolypeptide segments that assist in purification and/or detection of theantigen. Fusions can be made either at the amino terminus or at thecarboxy terminus of the antigen. Fusions may be direct with no linker oradapter molecule or may be through a linker or adapter molecule. Alinker or adapter molecule may be one or more amino acid residues,typically from about 20 to about 50 amino acid residues. A linker oradapter molecule may also be designed with a cleavage site for a DNArestriction endonuclease or for a protease to allow for the separationof the fused moieties. It will be appreciated that once constructed, thefused moieties may be derivatized or otherwise manipulated, as is knownin the art or according to the methods described herein. Suitable fusionsegments include, among others, metal binding domains (e.g., apoly-histidine segment), immunoglobulin binding domains (e.g., ProteinA, Protein G, T cell, B cell, Fc receptor, or complement proteinantibody-binding domains), sugar binding domains (e.g., a maltosebinding domain), and/or a “tag” domains (e.g., at least a portion ofα-galactosidase, a strep tag peptide, a T7 tag peptide, a FLAG peptide,or other domains that can be purified using compounds that bind to thedomain, such as monoclonal antibodies). This tag is typically fused tothe polypeptide upon expression of the polypeptide, and can serve as ameans for affinity purification of the sequence of interest polypeptidefrom the host cell. Affinity purification can be accomplished, forexample, by column chromatography using antibodies against the tag as anaffinity matrix. Optionally, the tag can subsequently be removed fromthe purified sequence of interest polypeptide by various means such asusing certain peptidases for cleavage.

In certain embodiments, the antigen may be directly or indirectly (e.g.,using an antibody) labeled or tagged in a manner which enables it to bedetected. An antigen may be directly labeled by attaching the label tothe antigen per se. An antigen may be indirectly labeled by attaching alabel to a reagent that binds to the antigen, such as an antibody orother moiety. Suitable labels include, for example, fluorochromes suchas fluorescein, rhodamine, phycoerythrin, Europium and Texas Red;chromogenic dyes such as diaminobenzidine, radioisotopes; macromolecularcolloidal particles or particulate material such as latex beads that arecoloured, magnetic or paramagnetic; binding agents such as biotin anddigoxigenin; and, biologically or chemically active agents that candirectly or indirectly cause detectable signals to be visually observed,electronically detected or otherwise recorded, for example in a FACS,ELISA, western blot, TRFIA, immunohistochemistry, evanescence, Luminexbead array, dipstick, or other lateral flow assay format. Suitableantibody-binding molecules for use in such methods may includeimmunoglobulin-binding antibodies, for example anti-human antibodies(e.g., anti-human antibodies specific for Ig isotypes or subclasses(e.g., of IgG), or specific for Staphylococcal protein A or G.

Preferred fluorescent tag proteins include those derived from the jellyfish protein known as green fluorescent protein (GFP). Furtherinformation on GFP and other fluorophores is given in the followingpublications: Tsien R Y, “The Green Fluorescent Protein” Annual Reviewsof Biochemistry 1998; 67:509-544 Verkhusha, V. and Lukyanov, K. “TheMolecular Properties and Applications of Anthoza Fluorescent Proteinsand Chromophores” Nature Biotechnology 2004; 22:289-296. Plasmid vectorsencoding a wide range of fluorescent tag proteins are commerciallyavailable from various suppliers including an array of “Living Colours8482; Fluorescent Proteins” available commercially from ClontechLaboratories, Inc. Similar vectors can also be obtained from othersuppliers including Invitrogen and Amersham Biosciences. Suitablefluorescent proteins derived from GFP are the red-shifted variant EGFP,the cyan shifted variant ECFP and the yellow shifted variant EYFP. EGFPis preferred as the fluorescent marker because it gives brightfluorescence combined with minimal effect on the antigenic properties ofthe target antigen. Alternative fluorescent marker proteins arecommercially available. Biologically or chemically active agents includeenzymes, which catalyse reactions that develop or change colours orcause changes in electrical properties, for example, and may also beutilized. They may be molecularly excitable, such that electronictransitions between energy states result in characteristic spectralabsorptions or emissions. They may include chemical entities used inconjunction with biosensors. Biotin/avidin or biotin/streptavidin andalkaline phosphatase detection systems may be employed. Further examplesinclude horseradish peroxidase and chemiluminescent reagents. In someembodiments, the non-immobilized antibody-binding molecule orpolypeptide may be detected using an antibody which binds to saidnon-immobilized antibody-binding molecule or polypeptide. A suitabledetection antibody may be labeled by means of fluorescence. In someembodiments, the label may be a fluorescent marker (tag) which is usedto label the target antigen directly such that the antigen and thefluorescent marker form a fusion protein.

If antibodies against the target antigen are present in a biologicalsample, the antigen or antibody may be labeled with a tag, and theantigen-antibody complex formed may be detected by, for example,immunoprecipitation. The fluorescence associated with the tag may thenbe used to determine that protein has been precipitated (qualitativedetermination) or to determine the amount of protein precipitated(quantitative determination). For example, soluble extracts of afluorescence-tagged antigen may be incubated with patient sera for anappropriate period of time such as overnight at 4° C. (typically 10-15μl of serum to 300-500 μl of extract or less) to allow antibodies tobind to the antigen. Protein A or Protein G Sepharose beads,preincubated with low IgG fetal calf serum (Sigma) to block non-specificbinding, are then added to the extract/serum mix containing the taggedantigen/antibody complexes, and mixed with gentle rotation for 1 to 2hours at room temperature. The antibodies within the serum, includingthose that specifically bind the tagged antigen, are bound by theprotein A/G beads. The protein A/G Sepharose beads are then washed in asuitable buffer (typically 10 mM Tris-HCl pH 7.4, 100 mM NaCl/ImMEDTA/1% Triton X-100) to remove any unbound tagged antigen. This may beachieved by multiple (e.g., 2, 3, 4, or 5) rounds of centrifugation,removal of the supernatant, and resuspension in buffer. The beads, somewith tagged antigen attached, are then collected and placed in afluorescence reader, for example a Spectra Max Gemini XS plate readerfrom Molecular Devices, Inc. The presence of antibodies in the samplemay then be quantitated. In the case of GFP, detection may beaccomplished using excitation at wavelength 472 nm and emission at 512nm. The fluorescence excitation will depend upon the fluorophore/tagthat is used but it would be possible to combine several differenttagged proteins in the same time. For example, one or more of Ply, PhtD,PhtE, LytB and/or PcpA may be separately tagged and separately orsimultaneously assayed. The sensitivity of the method is dependent onthe detection device and can be considerably enhanced by using moresensitive detection devices. Various modifications of these methodscould also be utilized. Other labels are available in the art and may besuitable for use the assays described herein.

The assays described herein for detecting antibodies immunoreactive withstreptococcal antigens may also be combined with other assays useful fordetecting streptococcal infection. For instance, these assays (i.e.,ELISA) may be combined with polymerase chain reaction (PCR) assays fordetecting streptococcal nucleic acid in a biological sample.Alternatively, an ELISA assay may be combined with animmunoprecipitation assay. Or, a PCR-based assay may be combined with animmunoprecipitation assay. Combining the various assays described hereinmay serve to even further increase the sensitivity of detection andfurther decrease the negative predictive value of the data.

Also provided herein as kits for detecting the presence of streptococcusinfection in a patient by detecting antibodies or nucleic acid in abiological sample of the patient. In one embodiment, one or morepneumococcal antigens (i.e., Ply, PhtD, PhtE, LytB, and/or PcpA) mayform part of a kit for detecting or diagnosing anti-streptococcalantibodies in a biological sample. The antigens may be provided in asuitable container such as a vial in which the contents are protectedfrom the external environment. Thus, a kit for detecting ananti-streptococcal antibody in a sample may comprise an antigen (i.e.,Ply, PhtD, PhtE, LytB, and/or PcpA) along with one or more detectionreagents for determining binding of one or more antibodies in a sampleto the antigen is provided. The kit preferably includes: (i) one or moreisolated and purified PcpA, PhtD, PhtE, LytB and/or PcpA proteins orimmunologically reactive fragments thereof of S. pneumoniae; and (ii)means for detecting the formation of an antigen-antibody complex,optionally packaged with instructions for use.

The antigen(s) may be free in solution or may be immobilized on a solidsupport, such as a magnetic bead, tube, microplate well, or chip. Incertain embodiments, a solid matrix comprising an isolated and purifiedPcpA, PhtD, PhtE, LytB, and/or PcpA protein or immunologically reactivefragment thereof or a fusion protein or protein aggregate adsorbedthereto is provided. In some embodiments, the kit may further comprisean antibody-binding molecule as a detection reagent. Theantibody-binding molecule may be a capture or detection reagent and maybe free in solution or may be immobilized on a solid support, such as amagnetic bead, tube, microplate well, or chip. The antibody-bindingmolecule or polypeptide may be labeled with a detectable label, forexample a fluorescent or chromogenic label or a binding moiety such asbiotin. Suitable labels are described in more detail above. The kit mayfurther comprise detection reagents such as a substrate, for example achromogenic, fluorescent or chemiluminescent substrate, which reactswith the label, or with molecules, such as enzyme conjugates, which bindto the label, to produce a signal, and/or reagents forimmunoprecipitation (i.e., protein A or protein G reagents). Thedetection reagents may further comprise buffer solutions, washsolutions, and other useful reagents. The kit may also comprise one orboth of an apparatus for handling and/or storing the sample obtainedfrom the individual and an apparatus for obtaining the sample from theindividual (i.e., a needle, lancet, and collection tube or vessel). Thekit may also include instructions for use of the antigen, e.g. in amethod of detecting anti-streptococcal antibodies in a test sample, asdescribed herein. Where the assay is to be combined with another type ofassay such as PCR, the required reagents for such an assay (i.e.,primers, buffers and the like) along with, optionally, instructions forthe use thereof, may also be included.

A better understanding of the present invention and of its manyadvantages will be had from the following examples, given by way ofillustration.

EXAMPLES Example 1 Materials and Methods

Ninety-nine children aged 2 months to 6 years were enrolled in aprospective cohort study at admission for CAP to the pediatric wards ofthe University Hospitals of Lausanne and Geneva (Switzerland) betweenMarch 2003 and December 2005. Children were eligible if presenting withclinical signs of pneumonia according to the WHO classification (ref),children with actively treated asthma, chronic illness or underlyingdisease, immunosuppression or presenting with wheezing (suspectedbronchiolitis) being excluded. None had been immunized against S.pneumoniae, according to the Swiss recommendation in 2003-2005. Theywere enrolled after signed parental content, as approved by the EthicalCommittee of both institutions.

Blood, urine and nasopharyngeal samples were cultivated within 8 hoursof admission and assessed for the presence of viruses or bacteria. PCRanalyses on nasopharyngeal samples included 13 viruses, Mycoplasmapneumoniae, and Chlamydia pneumoniae, whereas blood samples wereassessed by Ply-specific PCR. All chest radiographs taken at admissionwere reviewed by a senior radiologist blinded to clinical and laboratoryfindings. Convalescent serum samples were obtained 3 weeks later for75/99 children who had not been previously immunized against S.pneumoniae. These 75 children were included in this study onCAP-associated anti-pneumococcal responses.

The PhtD, PhtE, PcpA and LytB proteins used in this study arerecombinant proteins expressed in E. coli. The PhtD and PhtE used arefull-length proteins and the PcpA and LytB used are truncated forms withthe choline-binding domain removed. All four proteins are expressed inE. coli as soluble proteins and purified with combinations of ionexchange chromatography. All protein has ≧90% purity after purificationas assayed by SDS-PAGE and RP-HPLC.

Paired acute and convalescent serum samples were stored at −20° C. untilanalysis. Samples were encoded prior to transfer to the laboratory andIgG antibodies to Ply, PhtD, PhtE, PcpA and LytB were measured blindlyby laboratory personal unaware of clinical data. Paired serum sampleswere tested in the same run by indirect ELISA using purified proteins tocoat Immulon (Thermo Labsystem) plates. Eight serial dilutions of eachserum sample were performed to allow quantification of antibody titers.After 60 min at 37° C., anti-IgG antibody conjugated to horseradishperoxydase (Cappel) was added, followed by ABTS as the substrate. TheELISA titer of each serum was defined by comparison to a reference humanAB serum, used in each assay, to which ELISA Units were arbitrarilyassigned by taking the reciprocal of its dilution at OD=1.0. Resultswere expressed in EU/ml. Serum with titers below the assay cut-off of 5EU/ml were given a titer of 2.5 EU/ml. Antibody titers were logtransformed to allow comparisons of mean geometric concentrations (GMC).A significant rise in antibody titers was predefined as a minimaltwo-fold (100%) increase between the acute- and convalescent-phasesamples.

Socio-demographic characteristics of the participants are describedusing standard descriptive statistics (frequencies, means, geometricmeans, and standard deviation). Comparison of different serologies wereperformed using Student's T-test, while categorical data were comparedusing chi-square tests or Fisher's exact test when appropriate.Serologies among groups were compared by using analysis of variance(ANOVA). Univariate statistical analyses were performed for eachvariable to determine its relationship to the dependent variable, beinga case patient or not. Logistic regression analysis was used tocalculate adjusted odds ratio (OR) and 95% confidence interval (CI),controlling for any statistically significant demographic variables thatmight function as confounders (gender, etc). For all statistical tests,differences were considered significant at p<0.05 or when the 95% CI didnot include 1.0. SPSS (version 15.0) statistical-software program wasused for analyses.

Example 2 Experimental Results

A. Evidence of Acute Pneumococcal Infection in Children with Cap

Seventy-five previously healthy children (mean age 33.7 months, median35.4 months, range 2.6 to 66 months, 50% females) were enrolled atadmission and provided both acute and convalescent sera for thisprospective study of CAP-associated anti-pneumococcal immunity. Only onechild had a positive blood culture whereas 15/75 (20%) patients hadpneumolysin DNA (Ply⁺-PCR) in their blood. The use of serum IgGseroresponses to Ply (≧2-fold increase between acute and convalescentsera) identified 16/75 (21%) children with evidence of an acutepneumococcal infection (Table 1). This proportion increased to 31% (23/75) when combining Ply⁺-PCR and/or anti-Ply seroresponses, which isin accordance with recent reports (reviewed by Korppi M, Eur J ClinMicrobiol Infect Dis 2007).

To identify further children with evidence of acute pneumococcalinfection, we used four additional PSP (PhtD, PhtE, LytB, and PcpA) asimmunological probes to quantify antibody titers in the 75 paired acuteand convalescent serum samples. Responses to LytB were rare (Table 1).In contrast, significant (≧2-fold) IgG responses were observed in 21-32%of children hospitalized with CAP (Table 1). Altogether, 34/75 (45%)children had evidence of an acute response to S. pneumoniae. The meanfold-changes in serum IgG antibodies were marked for anti-PhtD (4.22),anti-PhtE (6.88) and anti-PcpA (5.62), moderate for anti-Ply (2.15) andweak for anti-LytB (1.51). Age did not influence the fold change ofserum anti-PSP antibodies (R²<0.162 for each PSP), indicating that evenyoung infants may raise anti-PSP responses to acute pneumococcalinfection. This was confirmed by the observation of three 8-10 monthsold infants with marked seroresponses (mean 2.88-6.82 fold changes) toPhtD, PhtE, and PcpA. Last, seroresponses were frequently directedagainst several PSP (≧2 PSP: 30%, ≧3 PSP: 25%, ≧4 PSP: 14%, ≧5 PSP: 1%),providing strong evidence of recent pneumococcal exposure.

The 34/75 (45%) children with acute seroresponses to ≧1 PSP included 86%( 13/15) Ply⁺-PCR patients. Only 2 Ply⁺-PCR children lacked anti-PSPresponse: a 2.6-month-old boy, presumably too young to rapidly raiseinfection-driven B cell responses, and a 43-month-old girl admitted witha 17-days history of cough, and 7 days of fever, who already had highacute serum titers against the 5 PSP when eventually admitted with lobarpneumonia. Thus, combining Ply⁺-PCR and seroresponses to a panel of 5PSP identified 36/75 (48%) children hospitalized for CAP as with strongevidence of acute pneumococcal infection (P-CAP).

B. Immunoprobes for the Diagnosis of Pneumococcal CAP in Young Children

The 36 CAP children with evidence of acute pneumococcal responses(≧2-fold rise) and/or infection (Ply⁺-PCR) (P-CAP) to 31 children withno evidence of recent pneumococcal exposure (negative Ply PCR and lackof a ≧2-fold rise of IgG titer to any PSP (NP-CAP) were studied. Eightchildren with very high (>300 EU/ml) admission serum titers against ≧3PSP were excluded from these analyses to avoid attribution errors, ashigh anti-PSP may reflect recent exposure or infection while limitingthe likelihood of a ≧2-fold response. Such an approach sets the assay'sspecificity and positive predictive value at 1.00, as responders are bydefinition not included in the control group. However, it allowscomparing assay sensitivity and negative predictive values inwell-controlled study groups. Relying on Ply⁺-PCR alone to diagnosepneumococcal CAP would have missed 19/36 (53%) patients, in accordancewith the fact that pneumococcal pneumonia is seldom bacteremic. The useof anti-Ply IgG responses alone would have missed 17/36 (47%) children,yielding a sensitivity of 0.44 and a negative predictive value of 0.61that are also in accordance with previous reports (Table 1). Highervalues resulted from the use of either anti-PhtE or anti-PcpA alone(Table 1). As postulated, combining several PSPs further increased assaysensitivity (Table 2). The combination of anti-PcpA and anti-PhtEresponses resulted in a sensitivity of 0.92, with a negative predictivevalue of 0.91. These values were further increased by adding anti-Plyresponses, the combination of anti-PcpA, PhtE and Ply responses yieldingthe optimal result of 0.94 for both sensitivity and negative predictivevalues. Importantly, the maximal sensitivity of any combination of PSPthat does not include PcpA remained below 0.68. This confirms thatincreasing the number of antigens enhances the likelihood of theidentification of a causal agent of CAP, and that certain immunoprobesmore significantly contribute to the diagnosis of pediatric pneumococcalCAP than others.

C. Clinical Characteristics of Children with Pneumococcal orNon-Pneumococcal CAP

It was then of interest to determine whether children with strongevidence of acute pneumococcal infection (P-CAP, n=36) differed in theirdemographic or clinical characteristics from children (n=31, NP-CAP)without evidence of recent pneumococcal exposure. Univariate analysesindicated that neither age (33.5 vs 32.7 months), gender (50% vs 48%females), clinical severity of pneumonia (WHO score I: 5 vs 1, II: 23 vs21, III: 7 vs 9), duration of cough (7 vs 4 days), duration of fever(4.2 vs 3.2 days) nor antibiotic use within 30 days of admission (9 vs 8children) differed in children hospitalized for P-CAP or NP-CAP. Thisconfirms that clinical patterns alone may not reliably identify childrenwith CAP of pneumococcal origin.

D. Distinct Pneumococcal Immunity in Children Admitted for Pneumococcalor Non-Pneumococcal CAP

Given the importance of preexisting anti-pneumococcal antibodies inprotection against S. pneumoniae infection, we used a common referenceserum to compare PSP-specific immunity at admission and possiblyidentify differences among P-CAP and NP-CAP children. All 75 patientshad detectable serum antibodies to at least one protein and most toseveral PSP (≧2 PSP: 96%, ≧3 PSP: 92%, ≧4 PSP: 89%, ≧5 PSP: 86%). Thehighest GMTs were directed against PcpA, Ply and PhtE, while anti-LytBantibodies were significantly lower (Table 3). Antibody titers atadmission were markedly heterogeneous. As both pneumococcal exposure andB cell response capacity increase with age, we searched for correlationsbetween age (months) and PSP-specific GMTs (log 10 EU/mL). Thesecorrelations were strong for Ply (R²=0.63434), PhtD (R²=0.63297) andPhtE (R²=0.59359) and weaker although significant for LytB (R²=0.45824)and PcpA (R²=0.31625). Assessing exposure-driven anti-PSP antibodies inchildren distributed in four even age groups (≦17, 18-35, 36-49 and ≧50months) indicated that anti-PcpA antibodies were already present at hightiters in children <18 months (FIG. 1), their further increase with agebeing significant (p=0.022) but less marked than for other PSPs(p<0.001). Anti-PcpA and anti-PhtE antibodies were already present inthe youngest infant, aged 2.6 months, presumably reflecting passivetransfer of maternal antibodies.

The five PSP used in this study are well conserved (>95-98%) amongpneumococcal strains. Should anti-PSP IgG antibodies play a role inprotection against pneumococcal pneumonia, one might therefore expect tofind differences in pneumococcal immunity between children withpneumococcal or non-pneumococcal CAP. At admission, antibodies to PhtD,PhtE and LytB were similar in P-CAP and NP-CAP children (Table 4 andFIG. 2), suggesting similar past exposure to S. pneumoniae and B cellresponse capacity. Anti-Ply at admission were significantly higher inP-CAP (446 EU/ml) than in NP-CAP (169 EU/ml, p=0.03) children. Thisdifference essentially reflected a greater proportion of P-CAP childrenwith high anti-Ply antibodies (>200 EU/ml; FIG. 2) at admission. Instriking contrast, anti-PcpA antibodies at admission were 3-fold lower(233 vs 716 EU/ml, p=0.001) in children hospitalized for P-CAP than forNP-CAP, reflecting a greater proportion of NP-CAP children withanti-PcpA titers at any value >10 EU/ml (FIG. 2). Thus, preexistingantipneumococcal immunity differed significantly between childrenhospitalized with CAP of pneumococcal and non-pneumococcal origin.

Convalescent antibody titers were then compared, harvested 3 weeks afteradmission for P-CAP or NP-CAP. Convalescent antibody titers to Ply,PhtD, PhtE and Lyt-B remained significantly (p<0.01) influenced by age,which in contrast did not influence anti-PcpA convalescent titers.Anti-LytB antibodies were not increased at all in convalescent sera,indicating the low infection-induced immunogenicity of this antigen(Table 4; FIG. 2). Convalescent antibody titers to the four other PSPwere higher in P-CAP than in NP-CAP patients. These differences onlyreached statistical significance for anti-Ply antibodies (Table 2). Thisis likely to essentially reflect the marked heterogeneity ofseroresponses in relatively small study groups as the reversedistributing cumulative curves clearly indicate the higher proportion ofmoderate or high anti-PhtD and anti-PhtE titers in P-CAP children (FIG.2). Interestingly, anti-PcpA antibodies which were 3-fold lower in P-CAPchildren at admission reached similar titers as in NP-CAP children only3 weeks after a single episode of pneumonia.

Multivariate analyses confirmed high Ply antibodies (p=0.014) and lowPcpA antibodies (p=0.004) at admission as the only significantpredictors of pneumococcal versus non-pneumococcal pneumonia inhospitalized children, regardless of their age and clinical scores.

E. Data Analysis

The data demonstrates that the use of a panel of immunogenic PSPssignificantly improves the diagnosis of pneumococcal infections in youngchildren with clinical signs of pneumonia, as defined by the WorldHealth Organization (WHO). WHO have identified anti-PcpA antibodies as akey diagnostic marker of pneumococcal CAP. These antibodies aredetectable at much lower levels during the acute phase in children withpneumococcal rather than non-pneumococcal pneumonia.

In this study, we postulated that the sensitivity of PSP-based assayscould be improved by the use of most immunogenic proteins and/or bytheir combination. The importance of PSP immunogenicity was largelyconfirmed by the demonstration of sensitivities ranging from 0.14 forLytB, to 0.44 for Ply, 0.56 for PhtD and 0.64 for either the PhtE orPcpA proteins. This ranking directly reflects the relativeimmunogenicity of PSP, as evidenced by a similar gradient of mean foldincrease of antibody responses (LytB (1.51), Ply (2.15), PhtD (4.22),PcpA (5.62) and PhtE (6.88)) largely independent of age. Moreimpressively, the combination of several PSP markedly increased assaysensitivity: combining anti-PcpA and anti-PhtE responses reached asensitivity of 0.92 for the diagnosis of a recent pneumococcalexposure/infection in children hospitalized for CAP, which was increasedto 0.94 by the addition of anti-Ply responses. One reason for theseimproved sensitivities, and their related negative predictive values, isthat past colonization or infections did usually not elicit antibodiesto all PSPs, such that high preexisting antibody titers to one PSP maynot prevent responses to other antigens. Another reason is theidentification of the PcpA protein as a key diagnostic marker ofpneumococcal CAP: in its absence, assay sensitivity reached 0.67 ratherthan 0.92 regardless of the PSP combination tested (Table 2).

The use of a panel of five PSP as immunoprobes identified 34/75 (45%)CAP children with evidence of acute pneumococcal responses. Thisincluded all patients with pneumococcal DNA in their blood (Ply⁺-PCR),with two exceptions: anti-PSP responses remained negative in a less than3 month-old infant hospitalized after a 4-day history of fever andcough. This suggests that serodiagnosis may remain difficult in veryyoung infants experiencing their first exposure to S. pneumoniae at timeof immune immaturity. As few young infants were enrolled in this study,this question will have to be addressed in subsequent studies. The otherPly⁺-PCR child lacking anti-PSP responses was a pre-schooler with aprolonged history of cough (17 days) and fever (7 days) prior toadmission for CAP. Her antibody titers were already very high athospitalization (anti-Ply: 952 EU/ml, anti-PhtD: 192 EU/ml, anti-PhtE:277 EU/ml, anti-PcpA: 462 EU/ml), indicating their activation prior toadmission. To note, the serodiagnosis of this patient would have beenconsidered as positive with less stringent study criteria than theexclusive use of fold-increase of antibody titers. Eight other children(mean age 44.5 months, range 22-66) had high preexisting immunity to ≧3PSP at admission (anti-Ply>380 EU/ml, anti-PhtD>111 EU/ml, anti-PhtE>393EU/ml and anti-PcpA>266 EU/ml), and were considered neither as P-CAP noras NP-CAP patients to avoid attribution errors. Respiratory viruses(RSV, hMPV, parainfluenza, rhinovirus, adenovirus, enterovirus) wereidentified in all eight except one, who had evidence of M. pneumoniaeinfection, and all Ply-PCR remained negative. Including these 8 patientsto the NP-CAP group would further increase the negative predictive valueof each anti-PSP assay.

It cannot be concluded that some anti-PSP responses resulted fromnasopharyngeal carriage. At admission, S. pneumoniae was more frequentlyidentified in the nasopharynx of P-CAP than NP-CAP patients (44% versus22%, p=0.06), in accordance with the fact that nasopharyngealacquisition precedes pneumococcal disease. For instance, it is knownthat the acquisition of a new pneumococcal strain induces thedevelopment of antibodies to certain PSP such as Ply, PhtB and PhtE(Holmlund E, PIDJ 2007). In contrast, pneumococcal carriage alone is notassociated with acute seroresponses. Nasopharyngeal sampling prior toadmission for CAP was not available to identify recent acquisition ofcarriage, and there are reasons to believe that anti-PcpA antibodies arenot readily elicited through nasopharyngeal carriage acquisition (seebelow). AOM was diagnosed at admission in three NP-CAP and four P-CAPpatients (NS), antibiotic prescription within 30 days being similar inboth groups. Previous prospective studies on the etiology of CAP did notinclude formal control groups of healthy children or of patientssuffering from other diseases. Interestingly, admission antibody titersof our 38 CAP patients aged 24-60 months (mean age 43.1 months,P-CAP:18, NP-CAP:18, indeterminate:2) were significantly lower thanthose of 58 healthy children (mean age 43.6 months) selected as controlswith no history of previous lower respiratory tract infection foranother study (Ply: 460 vs 745 EU/ml, PhtD: 150 vs 300 EU/ml, PhtE: 382vs 679 EU/ml, PcpA: 580 vs 1440 EU/ml, respectively). It will thus beinteresting to assess the influence of carriage acquisition, AOM andlower respiratory infections on PhtD, PhtE and PcpA immunity inprospective cohort studies.

The etiologic data presented herein corroborates well with findings ofother investigators. The attributed role of S. pneumoniae was indeeddocumented in 44% of cases in a US study with a similar design, studypopulation and extensive diagnostic workup (Michelow 2004). It is alsoin accordance with the 20-30% protective efficacy of a 7-valentpneumococcal conjugate vaccine. Should the high sensitivity andspecificity of these PSP-based immunoprobes be confirmed in othersettings, the same could thus prove extremely useful for the evaluationof the pediatric pneumococcal disease burden. Indeed, a pneumococcaletiology may not be solely derived from clinical symptoms, as confirmedagain here. Increasing the sensitivity of the diagnosis of pneumococcalCAP would also greatly reduce the size of the studies required todemonstrate pneumococcal vaccine efficacy in various country settings.

It was also observed that anti-pneumococcal immunity at admission wassignificantly different in children admitted with pneumococcal versusnon-pneumococcal CAP. PSP-specific antibodies were found in allchildren, over a wide range of concentrations reflecting age andpast-exposure. Admission antibody levels to PhtD, PhtE and Lyt-B weresimilar in both groups, supporting the claim that these children wereotherwise healthy children who had been previously exposed to S.pneumoniae, with the possible exception of the youngest infants in whomantibodies may have been of maternal origin. At admission, anti-Ply IgGantibodies were ≧2-fold higher in P-CAP children. This significantdifference essentially reflected a greater proportion of P-CAP childrenwith high anti-Ply antibodies (>200 EU/ml; FIG. 2) already at admission.Thus, Ply-specific responses were more rapidly induced in children withP-CAP than responses to other PSP. It is tempting to postulate thatanti-Ply immunity had been previously induced in these patients,allowing rapid anamnestic responses at time of pneumococcal infection.The observation of higher admission anti-Ply antibodies is in accordancewith the findings of others and supports the inclusion of “high”anti-Ply titers in the serologic diagnostic criteria of pneumococcalCAP.

In striking contrast, anti-PcpA antibodies were 3-fold lower in childrenadmitted for pneumococcal CAP, a difference which was highly significant(p=0.001). This is because saliva has the highest in vivo concentrationof Mn2+ (36 μM), such that PcpA expression is repressed unlesspneumococci invade the lung or bloodstream, where the levels of Mn2+ are1.000-fold lower (20 nM). As PcpA is not expressed during nasopharyngealcolonization, anti-PcpA responses reflect pneumococcal disease ratherthan colonization. This may contribute to the unique sensitivity of thePcpA-based assay (Table 3), which avoids confusion withnasopharyngeal-elicited responses. This PcpA expression pattern hasanother implication: children with preexisting anti-PcpA immunity arethose in whom pneumococcal disease has occurred previously. Conversely,low anti-PcpA antibody titers at time of admission for CAP indicate thatchildren may undergo a primary episode of pneumococcal disease—whichcould be associated with a higher risk of lower respiratory disease.PcpA antibodies may be just a marker of the protective immunity raisedby previous disease. In any case, PcpA appears to play a critical rolein establishing pneumococcal pneumonia and, therefore, needs to befurther assessed as a potential vaccine or diagnostic component.

In summary, a panel of five pneumococcal surface proteins (PSP) was usedto identify pneumococcal infection in a prospective study of 75 youngchildren (mean age 33.7 months) hospitalized with CAP. Twenty-three(31%) patients had either a positive pneumolysin (Ply) blood PCR (20%),or a ≧2-fold increase of anti-Ply antibodies (21%). Adding PhtD, PhtE,LytB and PcpA as immunological probes identified 36/75 (45%) patientswith acute pneumococcal infection (P-CAP), increasing the sensitivity ofthe diagnosis from 0.44 (Ply alone) to 0.94. Neither age, gender, WHOscores for clinical severity, duration of cough/fever or priorantibiotic use distinguished these 36 patients from 31 children with noevidence of recent pneumococcal exposure (NP-CAP). At admission,antibodies to PhtD, PhtE and Lyt-B were similar in both groups, whereasanti-Ply antibodies were significantly higher in P-CAP patients than inNP-CAP patients (446 vs 169 EU/ml, respectively; p=0.031). In contrast,P-CAP children had three-fold lower anti-PcpA antibodies (233 vs 716EU/ml, p=0.001). Multivariate analyses confirmed low PcpA antibodies attime of admission as the most significant predictor (p=0.004) of P-CAPin young children, in accordance with the preferential expression of PcpA in low Mn²⁺ compartments such as the lung rather than the nasopharynx.

All references cited herein are hereby incorporated by reference intheir entirety into this disclosure. While the present invention hasbeen described in terms of the preferred embodiments, it is understoodthat variations and modifications will occur to those skilled in theart. Therefore, it is intended that the appended claims cover all suchequivalent variations that come within the scope of the invention asclaimed.

TABLE 1 Convalescent responses to surface pneumococcal proteinsResponders (≧2-fold) Mean 95% CI Range (N/%) (EU/ml) (EU/ml) GMT (EU/ml)Sensitivity NPV Ply 16/75 (21%) 895 (389-1401) 154.33 ND-13079 0.44 0.61PhtD 20/75 (27%) 436 (147-723)  107.48 ND-10287 0.56 0.66 PhtE 24/75(32%) 978 (302-1654) 241.89 ND-24877 0.64 0.70 LytB 5/75 (7%) 37(25-49)  23.94 ND-439 0.14 0.50 PcpA 23/74 (31%) 1062 (668-1456) 281.04ND-10226 0.64 0.70 ND: below detection level; 95% CI: 95% Confidenceinterval; GMT: geometric mean titers NPV: negative predictive value

TABLE 2 Sensitivities of the combination of anti-PSP responses for thediagnosis of pneumococcal CAP Ply PhtD PhtE PcpA LytB Ply 0.44 0.58 0.670.86 0.47 PhtD 0.58 0.56 0.67 0.89 0.61 PhtE 0.67 0.67 0.64 0.92 0.67PcpA 0.86 0.89 0.92 0.64 0.72 LytB 0.47 0.61 0.67 0.67 0.14

TABLE 3 Exposure-driven serum IgG antibodies to surface pneumococcalproteins Seropositivity* GMT 95% CI Range N/% (EU/ml) (EU/ml) GMT(EU/ml) Ply 67/75 (89%) 498 (233-763) 110.59 ND-8790 PhtD 64/75 (85%)140 (109-171) 59.94 ND-604 PhtE 71/75 (95%) 326 (249-403) 130.03 ND-1561LytB 73/75 (97%) 30 (24-36) 21.79 ND-120 PcpA 69/74 (93%) 515 (375-655)148.36 ND-2563 ND: below detection level; 95% CI: 95% Confidenceinterval; GMT: geometric mean titers *Defined as ≧ 5 EU/ml

TABLE 4 Anti-PSP antibodies in children with CAP of pneumococcal versusnon-pneumococcal origin NP-CAP (n=31) P-CAP (n=36) P value Mean Mean (P-vs NP- PSP Stage (EU/ml) 95% CI GMT (EU/ml) 95% CI GMT CAP) Ply Acute169 (86-252) 58.34 446 (220-672)  115.8 0.031 PhtD Acute 125 (80-170)49.61 112 (74-150) 50.16 0.637 PhtE Acute 291 (190-392)  110.22 264(161-367)  104.26 0.712 PcpA Acute 716 (442-990)  241.67 233 (138-328) 66.64 0.001 LytB Acute 31 (21-41)  22.16 24 (18-30)  18.61 0.183 PlyConvalescent 165 (86-244) 58.82 1433 (409-2457) 235.13 0.023 PhtDConvalescent 132 (85-179) 49.39 725 (129-1321) 170.54 0.066 PhtEConvalescent 307 (195-419)  105.43 1615 (204-3026) 395.41 0.086 PcpAConvalescent 759 (456-1062) 247.13 1308 (534-2082) 247.98 0.216 LytBConvalescent 28 (20-36)  19.9 42 (17-67)  24.72 0.291 PSP: Pneumococcalsurface proteins NP-CAP: Community-Acquired Pneumonia without evidenceof acute pneumococcal infection P-CAP: Community-Acquired Pneumonia withevidence of acute pneumococcal infection 95% CI: 95% confidence intervalGMT: geometric mean liters

What is claimed is:
 1. A method of diagnosing pneumonia or an infectionby Streptococcus pneumoniae in a subject comprising: a) contacting abiological sample of the subject with PcpA, PhtD, PhtE, LytB and Plyantigens; and b) detecting complexes formed between antibodies in thebiological sample and at least two of the PcpA, PhtD, PhtE, LytB and Plyantigens; wherein detection of at least two complexes in step b) isindicative of infection by Streptococcus pneumoniae in the subject. 2.The method of claim 1 wherein detecting the formation of anantigen-antibody complex comprises detecting human immunoglobulin in theantigen-antibody complex.
 3. The method of claim 2 wherein detectinghuman immunoglobulin comprises contacting the antigen-antibody complexwith a second antibody that binds to human immunoglobulin for a time andunder conditions sufficient for said second antibody to bind to thehuman immunoglobulin in the complex and then detecting the boundanti-human immunoglobulin.
 4. The method of claim 2 wherein the secondantibody is labeled with a detectable marker or reporter molecule.
 5. Amethod for determining the response of a subject having pneumonia or aninfection by Streptococcus pneumoniae to treatment with a therapeuticcompound for said pneumonia or infection, said method comprising: a)contacting a biological sample of the subject with PcpA, PhtD, PhtE,LytB and Ply antigens; and b) detecting complexes formed betweenantibodies in the biological sample and at least two of the PcpA, PhtD,PhtE, LytB and Ply antigens; c) determining if the amount of thecomplexes detected is increased, unchanged or decreased in a biologicalsample of the subject obtained prior to treatment; wherein an unchangedor decreased amount of the complexes detected after treatment indicatesthat the subject is responding to treatment.
 6. The method of claim 5comprising contacting the biological sample with the antigens for a timeand under conditions sufficient for an antigen-antibody complex to formand then detecting the formation of an antigen-antibody complex.
 7. Themethod of claim 6 wherein detecting the formation of an antigen-antibodycomplex comprises detecting human immunoglobulin in the antigen-antibodycomplex.
 8. The method of claim 7 wherein detecting human immunoglobulincomprises contacting the antigen-antibody complex with a second antibodythat binds to human immunoglobulin for a time and under conditionssufficient for said second antibody to bind to the human immunoglobulinin the complex and then detecting the bound anti-human immunoglobulin.9. The method of claim 8 wherein the second antibody is labeled with adetectable marker or reporter molecule.
 10. The method of claim 6comprising performing an enzyme-linked immunosorbent assay (ELISA). 11.The method of claim 10 wherein the ELISA is a sandwich ELISA using acapture antibody and a detection antibody.
 12. A method of diagnosinginfection by Streptococcus pneumoniae in a subject comprising contactinga biological sample comprising an antibody with a solid matrixcomprising PcpA, PhtD, PhtE, LytB and Ply antigens under conditionssuitable for antibodies reactive to two or more of the antigens to bindthereto, and detecting the binding of said antibody to at least two ofsaid antigens, wherein detection of antibodies reactive to said antigensindicates the subject is infected by Streptococcus pneumoniae.